Chef gel blotting problem

Rafa Maldonado Rafael at genetics.med.utah.edu
Sat Feb 5 14:53:13 EST 1994


In article <msr2-040294183920 at cu-dialup-0221.cit.cornell.edu>
msr2 at cornell.edu (Mark S. Rose) writes:

> Hi folks
> Some people in our lab have been experiencing problems with blots of
> chromosomal  preparations run on CHEF gels.  Visible amounts of DNA in the
> form of reasonably well resolved chromosomes are present in our CHEF gels. 
> However when these gels are blotted to Hybond membranes and probed no
> signal is visible despite long exposure times (~1 week), use of very hot
> probes, and apparent absence of DNA in the gels after the transfer
> procedure.  For these blots we've been using our usual transfer procedure
> (1/2 hour in 0.2 N HCl, rince briefly in H2O, soak twice for 20 minutes
> each in 0.4M NaOH, 1M NaCl, transfer in 0.4M NaOH, 1M NaCl overnight, rinse
> blot briefly in H20, dry and bake).  Any suggestions would be appreciated.
> Thanks
> Mark

Hello Mark and everybody:
I don't know where the problem are, but your protocol is different in
several aspects that mine, and maybe these steps are important:
- I use a depurination step of 1 hour in 0.25 M HCl
- I always use a neutralization step (see Maniatis or whatever) after
the NaOH step.
- I transfer using SSC x20, and I never had problems. I think that
nitrocellulose doesn't like alkaline transfer, however Hybond is nylon,
right?
- Also, I never rinse the filters with H2O, I use SSC X2.
- I stick the DNA to the filters with UV. Anyway, I think sometimes I
have tried the oven and It worked.

Now, other questions:
Do you stain again with EtBr the gels to see if your DNA is still in
the gel? If not, you should, because thre is no EtBr in your gel after
an overnight transfer.
What's about your hybridization process? Perhaps your problem is there.
Do you include hybridization controls, like the plasmid of the probe,
in the filters?
Do you use 32P? with intensifier screens?
Some suggestions: use charged nylon or PVDF (from Millipore or
Amersham), which enhance the DNA binding to the membrane. Put more
amount of inserts in the wells. Try a  regular Southern (regular
chromosomal DNA extraction cut with some restriction enzyme) to see if
yor probe hybriduze with your DNA. 

By the way, what organism do you work in?

I hope I have been useful!

Rafa

----------------------------------
Rafael Maldonado
Howard Hughes Medical Insitute
University of Utah
Rafael at genetics.med.utah.edu



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